Summary Human leishmaniasis is a devastating infectious disease caused by protozoan parasites belonging to the genus of Leishmania. The disease is found in more than 90 countries and responsible for an estimated 1-2 million new infections each year worldwide. Leishmaniasis is also common in returning U.S. military personnel from Iraq and Afghanistan, with more than two thousand cases reported since 2001. Leishmania parasites cause disfiguring skin sores (cutaneous leishmaniasis or CL) and life-threatening infection of vital internal organs (visceral leishmaniasis or VL). VL (~20% of all leishmaniasis cases) is the most dangerous and fatal form of the disease, with a mortality rate close to 100% if left untreated. It is the second most deadly parasitic disease in the world (after malaria). Despite being a serious public health problem in many endemic regions, there are no vaccines or preventative chemotherapies available for leishmaniasis control. Current antileishmanial drugs have limited efficacy, serious side effects and high cost. Hence, there is a major unmet medical need for safe and effective drugs against leishmaniasis. Leishmanial CYP5122A1 is a novel cytochrome P450 (CYP) enzyme that is essential for the survival of L. donovani, a major causative agent for VL. Independent and our own studies have led us to hypothesize that identification of selective CYP5122A1 inhibitors as chemical probes will allow elucidation of its important role in the ergosterol biosynthesis by Leishmania. To test our hypothesis, three specific aims are proposed to answer three main questions: 1) How do the biochemical roles of CYP5122A1 and CYP51 compare in the ergosterol biosynthesis pathway of Leishmania? 2) Can selective CYP5122A1 inhibitors be identified and validated? and 3) Are arylimidamides (AIAs) and/or AIA-azole hybrids potential chemical probes to help delineate biochemical roles of CYP5122A1 in Leishmania biology? Innovative experimental approaches will be employed in the proposed project, e.g., a new HPLC-MS/MS-based sterol assay, fluorescence-based CYP5122A1 and CYP51 inhibition assays, and cutting-edge high content imaging-based intracellular antileishmanial assays. If successful, our proposed project to identify novel CYP5122A1 inhibitors will elucidate this protein?s role in Leishmania biology and validate CYP5122A1 as a drug target. Such an outcome could have a major positive impact on long-term leishmaniasis control around the globe.